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Background: New models continue to be required to improve our understanding of colorectal cancer progression. To this aim, we characterised in this study a three-dimensional multicellular tumour model that we named colospheres, directly obtained from mechanically dissociated colonic primary tumours and correlated with metastatic potential.Methods: Colorectal primary tumours (n=203) and 120 paired non-tumoral colon mucosa were mechanically disaggregated into small fragments for short-term cultures. Features of tumours producing colospheres were analysed. Further characterisation was performed using colospheres, generated from a human colon cancer xenograft, and spheroids, formed on agarose by the paired cancer cell lines.Results: Colospheres, exclusively formed by viable cancer cells, were obtained in only 1 day from 98 tumours (47%). Inversely, non-tumoral colonic mucosa never generated colospheres. Colosphere-forming capacity was statistically significantly associated with tumour aggressiveness, according to AJCC stage analysis. Despite a close morphology, colospheres displayed higher invasivity than did spheroids. Spheroids and colospheres migrated into Matrigel but matrix metalloproteinase (MMP)-2 and MMP-9 activity was detected only in colospheres. Mouse subrenal capsule assay revealed the unique tumorigenic and metastatic phenotype of colospheres. Moreover, colospheres and parental xenograft reproduced similar CD44 and CD133 expressions in which CD44+ cells represented a minority subset of the CD133+ population.Conclusion: The present colospheres provide an ex vivo three-dimensional model, potentially useful for studying metastatic process. [ABSTRACT FROM AUTHOR]

Plants are able to naturally graft or inosculate their trunks, branches and roots together, this mechanism is used by humans to graft together different genotypes for a range of purposes. Grafts are considered successful if functional vascular connections between the two genotypes occur. Various techniques can evaluate xylem connections across the graft interface. However, these methods are generally unable to assess the heterogeneity and three-dimensional (3D) structure of xylem vessel connections. Here we present the use of X-ray micro-computed tomography to characterize the 3D morphology of grafts of grapevine. We show that xylem vessels form between the two plants of natural root and human-made stem grafts. The main novelty of this methodology is that we were able to visualize the 3D network of functional xylem vessels connecting the scion and rootstock in human-made stem grafts thanks to the addition of a contrast agent to the roots and improved image analysis pipelines. In addition, we reveal the presence of extensive diagonal xylem connections between the main axial xylem vessels in 2-year old grapevine stems. In conclusion, we present a method that has the potential to provide new insights into the structure and function of xylem vessels in large tissue samples., (© 2024 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

In Alzheimer's disease (AD), the distribution of the amyloid precursor protein (APP) and its fragments other than amyloid beta, has not been fully characterized. Here, we investigate the distribution of APP and its fragments in human AD brain samples and in mouse models of AD in reference to its proteases, synaptic proteins, and histopathological features characteristic of the AD brain, by combining an extensive set of histological and analytical tools. We report that the prominent somatic distribution of APP observed in control patients remarkably vanishes in human AD patients to the benefit of dense accumulations of extra-somatic APP, which surround dense-core amyloid plaques enriched in APP-Nter. These features are accentuated in patients with familial forms of the disease. Importantly, APP accumulations are enriched in phosphorylated tau and presynaptic proteins whereas they are depleted of post-synaptic proteins suggesting that the extra-somatic accumulations of APP are of presynaptic origin. Ultrastructural analyses unveil that APP concentrates in autophagosomes and in multivesicular bodies together with presynaptic vesicle proteins. Altogether, alteration of APP distribution and its accumulation together with presynaptic proteins around dense-core amyloid plaques is a key histopathological feature in AD, lending support to the notion that presynaptic failure is a strong physiopathological component of AD., (© 2022 The Authors. Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

A main characteristic of sphingolipids is the presence of a very long chain fatty acid (VLCFA) whose function in cellular processes is not yet fully understood. VLCFAs of sphingolipids are involved in the intracellular traffic to the vacuole and the maturation of early endosomes into late endosomes is one of the major pathways for vacuolar traffic. Additionally, the anionic phospholipid phosphatidylinositol-3-phosphate (PtdIns (3)P or PI3P) is involved in protein sorting and recruitment of small GTPase effectors at late endosomes/multivesicular bodies (MVBs) during vacuolar trafficking. In contrast to animal cells, PI3P mainly localizes to late endosomes in plant cells and to a minor extent to a discrete sub-domain of the plant's early endosome (EE)/trans-Golgi network (TGN) where the endosomal maturation occurs. However, the mechanisms that control the relative levels of PI3P between TGN and MVBs are unknown. Using metazachlor, an inhibitor of VLCFA synthesis, we found that VLCFAs are involved in the TGN/MVB distribution of PI3P. This effect is independent from either synthesis of PI3P by PI3-kinase or degradation of PI(3,5)P 2 into PI3P by the SUPPRESSOR OF ACTIN1 (SAC1) phosphatase. Using high-resolution live cell imaging microscopy, we detected transient associations between TGNs and MVBs but VLCFAs are not involved in those interactions. Nonetheless, our results suggest that PI3P might be transferable from TGN to MVBs and that VLCFAs act in this process.

Little is known about the functional interaction between the Bloom's syndrome protein (BLM) and the recombinase RAD51 within cells. Using RNA interference technology, we provide the first demonstration that RAD51 acts upstream from BLM to prevent anaphase bridge formation. RAD51 downregulation was associated with an increase in the frequency of BLM-positive anaphase bridges, but not of BLM-associated ultrafine bridges. Time-lapse live microscopy analysis of anaphase bridge cells revealed that BLM promoted cell survival in the absence of Rad51. Our results directly implicate BLM in limiting the lethality associated with RAD51 deficiency through the processing of anaphase bridges resulting from the RAD51 defect. These findings provide insight into the molecular basis of some cancers possibly associated with variants of the RAD51 gene family.

Many cellular processes involve fast movements of weakly labeled cellular structures in all directions, which should be recorded in 3D time-lapse microscopy (4D microscopy). This chapter introduces fast 4D imaging, which is used for sampling the cell's volume by collecting focal planes in time-lapse mode as rapidly as possible, without perturbing the sample by strong illumination. The final images should contain sufficient contrast allowing for the isolation of structures of interest by segmentation and the analysis of their intracellular movements by tracking. Because they are the most sensitive, systems using wide-field microscopy and deconvolution techniques are discussed in greater depth. We discuss important points to consider, including system components and multifunctionality, spatial resolution and sampling conditions, and mechanical and optical stability and how to test for it. We consider image formation using high numerical aperture optics and discuss the influence of optical blur and noise on image formation of living cells. Spherical aberrations, their consequences for axial image quality, and their impact on the success of deconvolution of low intensity image stacks are explained in detail. Simple protocols for acquiring and treating point spread functions (PSFs) and live cells are provided. A compromise for counteracting spherical aberration involving the use of a kit of immersion oils for PSF and cell acquisition is illustrated. Recommendations for evaluating acquisition conditions and deconvolution parameters are given. Finally, we discuss future developments based on the use of adaptive optics which will push back many of today's limits.

Fluorescence labeling is the prevailing imaging technique in cell biology research. When they involve statistical investigations on a large number of cells, experimental studies require both low magnification to get a reliable statistical population and high contrast to achieve accurate diagnosis on the nature of the cells' perturbation. Because microscope objectives of low magnification generally yield low collection efficiency, such studies are limited by the fluorescence signal weakness. To overcome this technological bottleneck, we proposed a new method based on metal-coated substrates that enhance the fluorescence process and improve collection efficiency in epifluorescence observation and that can be directly used with a common microscope setup. We developed a model based on the dipole approximation with the aim of simulating the optical behavior of a fluorophore on such a substrate and revealing the different mechanisms responsible for fluorescence enhancement. The presence of a reflective surface modifies both excitation and emission processes and additionally reshapes fluorescence emission lobes. From both theoretical and experimental results, we found the fluorescence signal emitted by a molecular cyanine 3 dye layer to be amplified by a factor approximately 30 when fluorophores are separated by a proper distance from the substrate. We then adapted our model to the case of homogeneously stained micrometer-sized objects and demonstrated mean signal amplification by a factor approximately 4. Finally, we applied our method to fluorescence imaging of dog kidney cells and verified experimentally the simulated results.