Biology subjects

Beckett, A. J.

Publications and source records attributed to Beckett, A. J..

6 recordsLinked to original sources

bioRxiv2024

Nucleophosmin mutations lead to abnormal, but reversible, nucleoli architecture and aggregate formation - implications for NPM1-targeting therapies in AML

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.

cancer biology↗

bioRxiv2024

The Chromosome Periphery is an Essential Compartment of Oocyte Chromosomes

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.

cell biology↗

bioRxiv2024

Rules of engagement for condensins and cohesins guide mitotic chromosome formation

During mitosis, interphase chromatin is rapidly converted into rod-shaped mitotic chromosomes. Using Hi-C, imaging, proteomics and polymer modeling, we determine how the activity and interplay between loop-extruding SMC motors accomplishes this dramatic transition. Our work reveals rules of engagement for SMC complexes that are critical for allowing cells to refold interphase chromatin into mitotic chromosomes. We find that condensin disassembles interphase chromatin loop organization by evicting or displacing extrusive cohesin. In contrast, condensin bypasses cohesive cohesins, thereby maintaining sister chromatid cohesion while separating the sisters. Studies of mitotic chromosomes formed by cohesin, condensin II and condensin I alone or in combination allow us to develop new models of mitotic chromosome conformation. In these models, loops are consecutive and not overlapping, implying that condensins do not freely pass one another but stall upon encountering each other. The dynamics of Hi-C interactions and chromosome morphology reveal that during prophase loops are extruded in vivo at [~]1-3 kb/sec by condensins as they form a disordered discontinuous helical scaffold within individual chromatids.

molecular biology↗

bioRxiv2019

Comparison of BOLD and CBV using 3D EPI and 3D GRASE for cortical layer fMRI at 7T.

PurposeFunctional MRI (fMRI) at the mesoscale of cortical layers and columns requires both sensitivity and specificity, which can be compromised if the imaging method is affected by vascular artifacts, particularly cortical draining veins at the pial surface. Recent studies have shown that cerebral blood volume (CBV) imaging is more specific to the actual laminar locus of neural activity than BOLD imaging when using standard gradient-echo (GE) EPI sequences. Gradient and Spin Echo (GRASE) BOLD imaging has also shown greater specificity when compared with GE-BOLD.\n\nMethodsHere we directly compare CBV and BOLD contrasts in high-resolution imaging of the primary motor cortex for laminar fMRI in four combinations of signal labeling, VASO (CBV) and BOLD with 3D GE-EPI and zoomed 3D GRASE image readouts.\n\nResultsWe find that both CBV imaging using EPI-VASO and BOLD imaging using GRASE-BOLD, show similar specificity and sensitivity and are thus useful tools for mesoscopic fMRI in the human cortex.\n\nConclusionThese techniques demonstrate sufficient sensitivity and specificity to allow layer-fMRI to be used by neuroscientists in a wide range of investigations of depth-dependent neural circuitry in the human brain.

neuroscience↗

bioRxiv2019

Trypanosoma brucei colonises the tsetse gut via an immature peritrophic matrix in the proventriculus

The peritrophic matrix (PM) of haematophagus insects is a chitinous structure that surrounds the bloodmeal, forming a protective barrier against oral pathogens and abrasive particles. To establish an infection in the tsetse midgut, Trypanosoma brucei must colonise the ectoperitrophic space (ES), located between the PM and gut epithelium. Although unproven, it is generally accepted that trypanosomes reach the ES by directly penetrating the PM in the anterior midgut. Here we revisited this event by employing novel fluorescence and electron microscopy methodologies and found that instead, trypanosomes reach the ES via the newly secreted PM in the tsetse proventriculus. Within this model, parasites colonising the proventriculus can either migrate to the ES or become trapped within PM layers forming cysts that move along the entire gut as the PM gets remodelled. Early proventricular colonisation appears to be promoted by unidentified factors in trypanosome-infected blood, resulting in higher salivary gland infections and potentially increasing parasite transmission.

microbiology↗

bioRxiv2016

Microtubule organization within mitotic spindles revealed by serial block face scanning EM and image analysis

Serial block face scanning electron microscopy (SBF-SEM) is a powerful method to analyze cells in 3D. Here, working at the resolution limit of the method, we describe a correlative light-SBF-SEM workflow to resolve microtubules of the mitotic spindle in human cells. We present three examples of uses for this workflow which are not practical by light microscopy and/or TEM. First, distinguishing closely associated microtubules within K-fibers; second, resolving bridging fibers in the mitotic spindle; third, visualizing membranes in mitotic cells, relative to the spindle apparatus. Our workflow also includes new computational tools for exploring the spatial arrangement of MTs within the mitotic spindle. We use these tools to show that microtubule order in mitotic spindles is sensitive to the level of TACC3 on the spindle.

cell biology↗